Most mammalian cells cannot degrade cholesterol. When cellular cholesterol is no longer required as a metabolic intermediate for membrane stabilization, it must either be released from the cell or stored in the cytosol. Addition of long chain fatty acids to cholesterol via the esterification process reduces its solubility in the phospholipid bilayer and triggers its transfer to the cytoplasm where it is stored as liquid droplets (Rudel and Shelness, Nat. Med., 2000, 6, 1313–1314). Storage of cholesterol in droplets may serve to protect the cells from the toxicity of free cholesterol (Buhman et al., Biochim. Biophys. Acta, 2000, 1529, 142–154). In macrophages, the accumulation of cytosolic droplets of cholesterol esters results in the formation of foam cells in early atherosclerotic lesions (Buhman et al., Biochim. Biophys. Acta, 2000, 1529, 142–154).
Control of the risk factors involved in hypercholesterolemia and cardiovascular disease has been the focus of much research in academia and industry. Because an elevated level of circulating plasma low-density lipoprotein cholesterol has been identified as an independent risk factor in the development of hypercholesterolemia and cardiovascular disease, many strategies have been directed at lowering the levels of cholesterol carried in this atherogenic lipoprotein.
Acyl-CoA cholesterol acyltransferase (ACAT) enzymes catalyze the synthesis of cholesterol esters from free cholesterol and fatty acyl-CoA. These enzymes are also involved in regulation of the concentration of cellular free sterols (Buhman et al., Biochim. Biophys. Acta, 2000, 1529, 142–154; Burnett et al., Clin. Chim. Acta, 1999, 286, 231–242; Chang et al., Annu. Rev. Biochem., 1997, 66, 613–638; Rudel et al., Curr. Opin. Lipidol., 2001, 12, 121–127; Rudel and Shelness, Nat. Med., 2000, 6, 1313–1314).
Chang et al. cloned the first example of a human ACAT gene in 1993 (Chang et al., J. Biol. Chem., 1993, 268, 20747–20755). This original ACAT enzyme is now known as ACAT-1. Subsequently, the work of Meiner et al. suggested the presence of more than one ACAT gene in mammals (Meiner et al., J. Lipid Res., 1997, 38, 1928–1933). The cloning and expression of a second human ACAT isoform now known as acyl CoA cholesterol acyltransferase-2, was accomplished recently (Oelkers et al., J. Biol. Chem., 1998, 273, 26765–26771). Murine acyl CoA cholesterol acyltransferase-2 has also been identified and cloned (Cases et al., J. Biol. Chem., 1998, 273, 26755–26764).
Acyl CoA cholesterol acyltransferase-1 has multiple mRNA transcripts ranging from 1.9–7.2 kb that have been shown to be ubiquitously expressed. By contrast, the acyl CoA cholesterol acyltransferase-2 message is a single mRNA, approximately 2.5 kb, which is expressed predominately in the liver and intestine (Chang et al., J. Biol. Chem., 2000, 275, 28083–28092; Joyce et al., Curr. Opin. Lipidol., 1999, 10, 89–95).
The active site of acyl CoA cholesterol acyltransferase-1 is predicted to be cytoplasmic whereas acyl CoA cholesterol acyltransferase-2 is predicted to be on the lumenal side of the endoplasmic reticular membrane (Anderson et al., J. Biol. Chem., 1998, 273, 26747–26754).
In hepatocytes, cholesterol esters along with triacylglycerols constitute the bulk of the neutral lipid core of very low density lipoprotein (VLDL) (Chang et al., J. Biol. Chem., 2000, 275, 28083–28092). Based on the hypothesis that inhibitors of ACAT enzymes can lower plasma cholesterol levels, considerable research efforts have focused on the discovery of small molecule inhibitors of ACAT enzymes as cholesterol-lowering and/or anti-atherosclerotic agents. This field has been reviewed recently (Burnett et al., Clin. Chim. Acta, 1999, 286, 231–242; Chong and Bachenheimer, Drugs, 2000, 60, 55–93; Davignon, Diabete Metab., 1995, 21, 139–146; Krause and Bocan, ACAT inhibitors: physiologic mechanisms for hypolipidemic and antiatherosclerotic activities in experimental animals. In Inflammation: Mediators and Pathways. Eds. Ruffalo, R. R Jr. and Hollinger, M. A. pp 173–197, 1995, CRC Press, Boca Raton; Matsuda, Med. Res. Rev., 1994, 14, 271–305; Roth, Drug Discovery Today, 1998, 3, 19–25). A partial list of classes of small molecule inhibitors of ACAT enzymes includes: fatty acyl amides (Krause and Bocan, ACAT inhibitors: physiologic mechanisms for hypolipidemic and antiatherosclerotic activities in experimental animals. In Inflammation: Mediators and Pathways. Eds. Ruffalo, R. R Jr. and Hollinger, M. A. pp 173–197, 1995, CRC Press, Boca Raton; Roth, Drug Discovery Today, 1998, 3, 19–25), substituted ureas (Tanaka et al., J. Med. Chem., 1998, 41, 4408–4420; Tanaka et al., Bioorg. Med. Chem., 1998, 6, 15–30; Tanaka et al., J. Med. Chem., 1998, 41, 2390–2410) sulfamates (Bocan et al., Arterioscler. Thromb. Vasc. Biol., 2000, 20, 70–79; Nicolosi et al., Atherosclerosis, 1998, 137, 77–85), sulfonamides (Lee et al., Bioorg. Med. Chem. Lett., 1998, 8, 289–294), acyl phosphonamides (Lee et al., Bioorg. Med. Chem. Lett., 1998, 8, 289–294), acyl phosphoroamadites (Lee et al., Bioorg. Med. Chem. Lett., 1998, 8, 289–294), phosphonates (Sellers et al., Toxicol. Sci., 1998, 46, 151–154), phenylethylamines (Dugar et al., Bioorg. Med. Chem., 1995, 3, 1231–1236; Vaccaro et al., J. Med. Chem., 1996, 39, 1704–1719), bioflavinoid derivatives (Lee et al., Ann. Nutr. Metab., 1999, 43, 173–180), heterocyclic amides (White et al., J. Med. Chem., 1996, 39, 3908–3919) and tetrazole-amide derivatives (O'Brien et al., J. Med. Chem., 1996, 39, 2354–2366).
There are ongoing clinical studies with small molecule ACAT inhibitors but preliminary reports suggest poor gastrointestinal tract tolerability in humans (Chong and Bachenheimer, Drugs, 2000, 60, 55–93).
Disclosed and claimed in PCT publication WO 99/67368 is the nucleic acid sequence encoding acyl CoA cholesterol acyltransferase-2 and methods for modulating a symptom, in a mammalian host, of a disease condition associated with acyl CoA cholesterol acyltransferase-2 activity, said method comprising an effective amount of an active agent that modulates or selectively inhibits said acyl CoA cholesterol acyltransferase-2 activity in said host (Cases et al., 1999). Disclosed and claimed in Japanese patent JP 6-172186 is an inhibitor containing, as active ingredient(s), at least one pyrimidine base, purine base, and nucleoside with the above base(s) as the constituent(s) wherein said inhibitor is useful for the prevention and treatment of various diseases involving arteriosclerosis (Shohachi, 1994).
Currently, inhibitors of ACAT enzymes include several classes of non-isozyme-specific small molecules. Consequently, there remains a long felt need for additional agents capable of effectively and selectively inhibiting the function of acyl CoA cholesterol acyltransferase-2.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of expression of acyl CoA cholesterol acyltransferase-2.
The present invention provides compositions and methods for modulating expression of acyl CoA cholesterol acyltransferase-2.